Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

Resistin induces insulin resistance in pancreatic islets to impair glucose-induced insulin release

An adipokine resistin, a small cysteine-rich protein, is one of the major risk factors of insulin resistance. In the present study, transiently resistin-expressing mice using adenovirus method showed an impaired glucose tolerance due to insulin resistance. We found that resistin-expressing mice exhibited impaired insulin secretory response to glucose. In addition, in vitro treatment with resistin for 1 day induced insulin resistance in pancreatic islets and impaired glucose-stimulated insulin secretion by elevating insulin release at basal glucose (2.8 mM) and suppressing insulin release at stimulatory glucose (8.3 mM). In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets as well as other insulin target organs. Furthermore, resistin induced SOCS-3 expression in beta-cells. In conclusion, resistin induces insulin resistance in islet beta-cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation and impairs glucose-induced insulin secretion.     

Lipoxygenase-generated icosanoids inhibit glucose-induced insulin release from rat islets

Lipoxygenase-pathway metabolites of arachidonic acid are produced in pancreatic islets. They are are implicated in insulin release, since nonselective inhibitors of lipoxygenases inhibit glucose-induced insulin release. We studied the interplay in insulin release between glucose and selected icosanoids formed in 5-, 12- and 15-lipoxygenase pathways. Effects on immunoreactive insulin release of 10(7) to 10(6)-12-(R)-HETE, 12-(S)-HETE, hepoxilin A3, lipoxin B4, LTB4 or LTC4 were tested individually in 30-min incubations of freshly isolated young adult Wistar rat pancreatic islets, in the presence of 5.6 mM or 23 mM glucose. Basal insulin release (at 5.6 mM glucose) was stimulated by LTC4 and hepoxilin A3 (304% and 234% of controls at 5.6 mM glucose alone, respectively), inhibited by 12-(S)-HPETE (56%), and was not affected by 12-(R)-HETE, 12-(S)-HETE, lipoxin B4 or LTB4 (111%, 105%, 106% and 136%, respectively). Insulin release evoked by 23 mM glucose (190-320%) was inhibited (50-145%) by all icosanoids tested, except LTC4 (162%). We conclude that, among the lipoxygenase products tested, only leukotrienes and hepoxilin are candidates for a tonic-stimulatory influence on basal insulin release. Since glucose promotes icosanoid formation in islets, the observed inhibition of glucose-induced insulin release by lipoxygenase products suggests the existence of a negative-feedback system.     

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

Characteristics of the biotin enhancement of glucose-induced insulin release in pancreatic islets of the rat

Perifused isolated rat islets were used to show that biotin plus 16.5 mM glucose evoked more insulin secretion than 16.5 mM glucose alone. Whether or not this reinforcement of glucose-induced insulin secretion by biotin is unique was studied by using perifused islets stimulated with 16.5 mM glucose plus 100 microM of one of various components of the vitamin B group. No effect of any of these vitamins was found on glucose-induced insulin secretion. These results indicate that biotin is unique among the members of the vitamin B group in enhancing glucose-induced insulin secretion. Static incubation experiments showed that biotin did not potentiate insulin release when the islets were incubated with an experimental solution containing either no or 2.8 mM glucose. The addition of biotin to 27.7 mM glucose, which is the maximal concentration for stimulating insulin release, did not significantly enhance the effect of the glucose on insulin release (although it did at 16.5 mM glucose). These findings indicate that biotin, by itself, does not stimulate insulin secretion, and does not enhance glucose-induced insulin secretion beyond the ability of glucose itself to stimulate insulin secretion.     

Stimulation of insulin release from pancreatic islets by quinones

Coenzyme Q (CoQ₀) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ₀benzoquinonehydroquinonemenadione. CoQ₆ and CoQ₁₀ (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ₀'s insulinotropism was enhanced in calcium-free medium and CoQ₀ appeared to stimulate only the second phase of insulin release. CoQ₀ inhibited inositol mono-, bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ₀-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ₀-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occuring quinones, such as CoQ₁₀, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis.     

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion.

The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.3 mM-glucose, 0.43 mM-Ca2+) abolished TPA (0.16 microM)-induced insulin secretion and led to a potentiation of phase 1 and a decrease in phase 2 of glucose-induced insulin secretion. Thus, although the total insulin release during 40 min of perfusion with glucose (16.7 mM) (45-85 min) was unaffected, the percentage released during phase 1 (45-55 min) was increased from 12.9 +/- 1.5 (4)% in controls to 35.8 +/- 3.9 (4)% in TPA-treated islets (P less than 0.01), and the percentage released during phase 2 (65-85 min) was decreased from 63.2 +/- 3.9 (4)% to 35.3 +/- 1.4 (4)% (P less than 0.005). In contrast, TPA exposure in TCM 199 medium (5.5 mM-glucose, 1.26 mM-Ca2+) caused a total abolition of both phases 1 and 2 of glucose-induced secretion. However, inclusion of the alpha 2-adrenergic agonists adrenaline (10 microM) or clonidine (10 microM), or lowering of the Ca2+ concentration in TCM 199 during down-regulation, preserved and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1 and inhibited phase 2 of carbamoylcholine (100 microM)-induced insulin secretion at 3.3 mM-glucose, and abolished the potentiating effect of carbamoylcholine (100 microM) at 16.7 mM-glucose. These results substantiate a role for protein kinase C in insulin secretion, and suggest that protein kinase C inhibits phase 1 and stimulates phase 2 of both glucose-induced and carbamoylcholine-induced insulin secretion.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Involvement of novel protein kinase C isoforms in carbachol-stimulated insulin secretion from rat pancreatic islets

The roles of protein kinase C (PKC) isoforms in cholinergic potentiation of glucose-induced insulin secretion were investigated in rat pancreatic islets. Western-blot analysis showed the presence of PKC-alpha, betaII, delta, epsilon, eta, and zeta, but not PKC-betaI, gamma, or iota, in the islets. Carbachol (CCh) caused translocations of PKC-alpha, betaII, delta, and epsilon from the cytosol to the plasma membrane. CCh facilitated 7-mM glucose-induced insulin secretion from isolated rat islets. The CCh-stimulated insulin secretion was significantly suppressed by the generic PKC inhibitor chelerythrine. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, had no effect on the insulin secretion stimulated by CCh, although it significantly inhibited that induced by phorbol 12-myristate 13-acetate. These results suggest that the novel PKC isoforms activated by CCh, i.e., PKC-delta and/or epsilon, participate in the stimulatory effect of CCh on insulin secretion.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

Stimulus-secretion coupling of glucose-induced insulin release. LI. Divalent cations and ATPase activity in pancreatic islets

Pancreatic islets can be viewed as a fuel-sensor organ. The amount of ATP used by the islet cells for the maintenance of adequate Ca2+ gradients across membranes is not known. An indirect approach to this issue consists in the measurement of Ca-ATPase activity. The kinetics of Ca-ATPase in islet homogenates yielded a Km for ATP close to 0.1 mM and two Km values for Ca2+ close to 0.13 and 4-6 microM, respectively. Within limits, the Ca-ATPase appeared as a distinct entity from Mg-ATPase. Several divalent cations, including Mg2+, inhibited the Ca-ATPase activity. Calmodulin also inhibited, significantly albeit modestly Ca-ATPase. The activity of the enzyme was increased at high pH or in the presence of bicarbonate. The reaction velocity at close-to-physiological concentrations of ATP, Ca2+ and H+ suggests that the consumption of ATP by the Ca-ATPase may account for a major fraction of the overall rate of ATP breakdown in intact islets.     

Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets.

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets.

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.     

Protein kinase C isoform expression and activity in the mouse heart

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.